1. Field of the Invention
The present invention relates to a fluorescence spectroscopy of analyzing a fluctuation in fluorescent molecules in a biological sample to analyze the state of the fluorescent molecules, and a fluorescence spectroscopic apparatus that analyzes the interaction between different fluorescent molecules.
2. Description of the Related Art
Fluorescence correlation spectroscopy (FCS) is a technique for analyzing a fluctuation in light caused by Brownian motion of fluorescent molecules in a fine observation area in a microscopic visual field to determine the autocorrelation function of fluorescence intensity, thus analyzing the diffusion time and average molecular weight for each molecule. Fluorescence correlation spectroscopy is described in, for example, Document 1.
Document 1: “One-Molecule Detection Based on Fluorescence Correlation Spectroscopy” Kinjyō, Protein, Nucleic Acid, and Enzyme, 1999, vol. 44, NO 9, 1431-1438.
Here, when the fluorescence intensity is defined as I(t), the autocorrelation function C(τ) is expressed by Equation (1).
                              c          ⁡                      (            τ            )                          =                              〈                                          I                ⁡                                  (                  t                  )                                            ⁢                              I                ⁡                                  (                                      t                    +                    τ                                    )                                                      〉                                              〈                              I                ⁡                                  (                  t                  )                                            〉                        2                                              Equation        ⁢                                  ⁢                  (          1          )                    
FIG. 5 is a diagram showing a measuring optical system used for such fluorescence correlation spectroscopy (FCS).
A laser is used as an excitation light source 101 that excites a sample. Laser light from the excitation light source 101 is reflected by a dichroic mirror 102 and enters an objective lens 103. A sample 104 labeled with a fluorescent dye is placed at the focal position of the objective lens 103. Laser light condensed in the focal portion of the objective lens 103 excites the fluorescent dye in the sample to induce fluorescence.
Fluorescence emitted by the fluorescent dye in the sample 104 reaches the dichroic mirror 102 via the objective lens 103. The dichroic mirror 102 has the optical characteristic that it reflects excitation light and allows fluorescence to pass through. The fluorescence from the sample 104 passes through the dichroic mirror 102 and is condensed by a condensing lens 105.
A pinhole 106 is located at the focal position of the condensing lens 105. The pinhole 106 blocks fluorescence from the objective lens 103 except for its focal position to achieve a high space resolution. Those of the fluorescences having passed through the pinhole 106 which are in a desired wavelength band, pass through a barrier filter 108 and enter a photodetector 109. The photodetector 109 measures a fluctuation in fluorescence intensity.
Fluorescence cross-correlation spectroscopy (FCCS) has been proposed which is obtained by expanding such fluorescence correlation spectroscopy (FCS). Fluorescence cross-correlation spectroscopy (FCCS) is a technique for determining the cross-correlation function between fluorescence signals to analyze the association between the signals. Fluorescence cross-correlation spectroscopy (FCCS) is used to, for example, analyze the interaction between molecules labeled with fluorescent dyes in two colors. Fluorescence cross-correlation spectroscopy (FCCS) is described in, for example, Documents 2 and 3 in detail.
Document 2: Dual-Color Fluorescence Cross-Correlation Spectroscopy for Multicomponent Diffusional Analysis in Solution, Petra, Schwille et al, Biophyiscal Journal 1997, 72, 1878-1886.
Document 3: A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy, Petra, Schwille et al, Methods 29 (2003) 74-85.
Here, two fluorescent dyes are defined as A and B and their fluorescence intensities are defined as IA(t) and IB(t), respectively. A cross-correlation function g(τ) is expressed by Equation (2).
                              g          ⁡                      (            τ            )                          =                              〈                                                            I                  A                                ⁡                                  (                  t                  )                                            ⁢                                                I                  B                                ⁡                                  (                                      t                    +                    τ                                    )                                                      〉                                              〈                                                I                  A                                ⁡                                  (                  t                  )                                            〉                        ⁢                          〈                                                I                  B                                ⁡                                  (                  t                  )                                            〉                                                          Equation        ⁢                                  ⁢                  (          2          )                    
Confocal fluorescence coincidence analysis (CFCA) has also been proposed which detects the coincidence of fluorescence fluctuations in two fluorescent molecules. This technique is described in, for example, Document 4.
Document 4: Confocal fluorescence coincidence analysis (CFCA), Winkler et al., Proc. Natl. Acad. Sci. U.S.A. 96:1375-1378, 1999.
This technique expresses a K value indicating the coincidence by Equation (3).
                              K          ⁡                      (            n            )                          =                                                            ∑                m                            ⁢                                                                    N                    1                                    ⁡                                      (                    m                    )                                                  ⁢                                                      N                    2                                    ⁡                                      (                    m                    )                                                                                                      ∑                m                            ⁢                                                                    N                    1                                    ⁡                                      (                    m                    )                                                  ⁢                                                      ∑                    m                                    ⁢                                                            N                      1                                        ⁡                                          (                      m                      )                                                                                                    *          n                                    Equation        ⁢                                  ⁢                  (          3          )                    
Much attention has been paid to non-aggressive, real-time measurements of these fluorescence spectroscopic methods (FCS, FCCS, and CFCA). In recent years, these methods have been used not only for solutions but also for various biological samples such as cells.
Fluorescence correlation spectroscopy (FCS) detects the presence of interaction on the basis of a variation in diffusion time and is unsuitable for reaction such as the interaction between proteins which does not exhibit a significant variation in diffusion time. On the other hand, fluorescence cross-correlation spectroscopy (FCCS) and confocal fluorescence coincidence analysis (CFCA) are not subject to such a limitation and are thus particularly expected to be applied to analysis of the interaction between proteins.
FIG. 6 is a diagram showing an optical system used for measurements for fluorescence cross-correlation spectroscopy (FCCS) and confocal fluorescence coincidence spectroscopy (CFCA). Optical systems similar to that shown in FIG. 6 are described in, for example, Non-Patent Documents 2, 3, and 4. These measuring optical system use two laser light sources as an excitation light source. For example, a blue laser (wavelength=488 nm) is used as an excitation light source 121. A green laser (wavelength=543 nm) is used as an excitation light source 122.
Laser light from the excitation light sources 121 and 122 is mixed into a single light flux by a dichroic mirror 124. The light flux is reflected by a dichroic mirror 125 and then enters an objective lens 126. A sample 127 is placed at the focal position of the objective lens 126; the sample 127 contains two types of molecules labeled respectively with a fluorescent dye A that is excited by blue excitation light and a fluorescent dye B that is excited by green excitation light.
Fluorescence emitted by the two fluorescent dyes in the sample 127 reaches the dichroic mirror 125 via the objective lens 126. The dichroic mirror 125 has the optical characteristic that it reflects excitation light and allows fluorescence to pass through. The fluorescence from the sample 127 passes through the dichroic mirror 125 and is condensed by a condensing lens 128. A pinhole 129 serves to achieve a high space resolution.
The fluorescence is subsequently separated by a dichroic mirror 131 into the fluorescence emitted by the fluorescent dye A and the fluorescence emitted by the fluorescent dye B. Only the fluorescences that are in desired wavelength bands pass through a barrier filter 133 (for example, passband: 495 to 535 nm) and a barrier filter 134 (for example, passband: 570 to 610 nm). These fluorescences enter photodetectors 135 and 136, respectively. The photodetectors 135 and 136 measure a possible fluctuation in fluorescence intensity.